The conjugation of proteins with ubiquitin involves the sequential action of proteins known as E1 (ubiquitin activating enzyme), E2s or UBCs (ubiquitin conjugating enzymes), and E3s (ubiquitin protein ligases). We have previously determined that RING finger proteins are in general ubiquitin ligases and have now focused on understanding issues related to the mechanism of action and substrates of specific members of this family as well as identifying inhibitors. Yeast two-hybrid screens carried out with Myriad Genetics have yielded a number of interesting interaction partners for E3 that include AO7 (RNF25), Ariadine and BRAP (IMP). For AO7 several of these interactions have been confirmed using a far-western approach, and in collaboration with Dr. Robert Coffey we have determined that at least one interacting protein, Naked-2 is a bona fide substrate for AO7. Further studies are currently ongoing to evaluate potential substrates for this and other E3s. Our studies looking for inhibitors of Mdm2 have resulted in the identification of a family of compounds that inhibit Mdm2 E3 activity both in vitro and in cells (HLI98 family) and we have now identified a related small molecule that is highly soluble in water and exhibits greater potency than the HLI98 compounds. This small molecule selectively kills p53-expressing cancer cells in a manner similar to the original compounds. Additionally, our screens have also resulted in the identification of an inhibitor of the ubiquitin E1 inhibitor. This inhibitor markedly deceases ubiquitin-mediated degradation of substrates as well as non-proteolytic functions of the ubiquitin system including ligand-induced activation of NfkB and ligand-mediated ubiquitination of the epidermal growth factor receptor. It also results in increased levels of p53 and in selective cell death in transformed cells expressing wild type p53. Thus, this new tool should be of great value for experimental purposes and may also serve as the basis for new therapeutics for cancer and other diseases in which perturbing functions of the ubiquitin system may be of beneficial. We are also carrying out structure-function and structural studies on the transmembrane ERAD ubiquitin ligase gp78. Preliminary results show good correlations between findings obtained from NMR (in collaboration with Dr. R. Andrew Byrd) and biochemical studies being carried out in our laboratory. These studies should provide new insights into the function of ubiquitin ligases. In addition to this we have been carrying out structure-function studies for the HRD1 ubiquitin ligase of yeast. In particular we have been focusing on an essential component of this ligase complex known as Cue1p. We have determined that a discrete region within Cue1p binds to the yeast ERAD E2 (Ubc7p) and is essential for ERAD in yeast. Interestingly the CUE domain of Cue1p appears to be dispensible. These studies in yeast complement and inform our studies in mammalian cells.